The invention relates generally to the field of mutant proteins and genes involved in cancer, and to the detection, diagnosis and treatment of cancer.
Cancer is major cause of death in humans. Lung cancer is a major cause of cancer-related mortality worldwide and is expected to remain a major health problem for the foreseeable future. It is broadly divided into small cell lung cancer (SCLC, 20% of lung cancers), and non-small cell lung cancer (NSCLC, 80% of lung cancers). Somatic mutations in the epidermal growth factor receptor (EGFR) gene are found in a subset of lung adenocarcinomas and are associated with sensitivity to the EGFR tyrosine kinase inhibitors (TKI) Gefitinib [Lynch, T. J., et al., N Engl J Med, 2004. 350(21): p. 2129-39, and Paez, J. G., et al., Science, 2004. 304(5676): p. 1497-500] and Erlotinib [Pao, W., et al., Proc Natl Acad Sci USA, 2004. 101(36): p. 13306-11]. Many types of EGFR mutations have been reported, but the most common non-small cell lung cancer (NSCLC)-associated EGFR mutations are the 15-bp nucleotide in-frame deletion in exon 19 (E746-A750del) and the point mutation replacing leucine with arginine at codon 858 in exon 21 (L858R) [Pao, W., et al., Proc Natl Acad Sci USA, 2004. 101(36): p. 13306-11; Riely, G. J., et al., Clin Cancer Res, 2006. 12(24): p. 7232-41; and Kosaka, T., et al., Cancer Res, 2004. 64(24): p. 8919-23. These two mutations represent 85-90% of EGFR mutations in NSCLC patients. Importantly, patients with these mutations have been shown to respond well to EGFR inhibitors including Gefitinib and Erlotinib [Riely, G. J., et al., Clin Cancer Res, 2006. 12(24): p. 7232-41; Inoue, A., et al., J Clin Oncol, 2006. 24(21): p. 3340-6; Marchetti, A., et al., J Clin Oncol, 2005. 23(4): p. 857-65; and Mitsudomi, T., et al., J Clin Oncol, 2005. 23(11): p. 2513-20.]. Therefore detection of these mutations is an important method to improve treatment of lung cancer patients.
Since EGFR mutational analysis in lung adenocarcinoma can guide treatment decisions and to enroll patients on specific arms of clinical trials, direct DNA sequencing of PCR amplified products has been developed to detect EGFR mutation in patient tumor tissue. However, these tests have not been widely adopted due the high costs of the equipment and reagents, the difficulty of performing the assay and the length of time required for completion of the test. In addition, DNA sequencing has a limited sensitivity for the detection of tumor cells containing an EGFR mutation within a background of nonmutant normal cells. A minimum of 50% tumor cells is required to ensure the accuracy of the EGFR sequencing assay. Recently, other DNA based methods have been developed to improve the detection of EGFR mutation in lung cancer specimens, including TaqMan PCR, Scorpions ARMS, MALDI TOF MS-based genotyping, dHPLC, and single molecule sequencing. However, these methods are not routine procedures in clinical labs and remain expensive and time-consuming. Also they do not identify mutation-status on a cellular basis. Therefore, their sensitivity is dependent on the percentage tumor cells contained in the sample used to produce the homogenate, and samples obtained from standard biopsy are usually not sufficient for DNA sequencing. On the other hand, Immunohistochemistry (IHC) is a well-established method of solid tumor analysis routinely performed in all clinical laboratories. This method is a more accessible technique in clinical diagnosis and the interpretation is less affected by the percentage of the cancer cells in the tumor specimens or the amount of tumor tissue available for analysis. The method also allows for the simultaneous analysis of other proteins or protein modifications. However, total expression level of EGFR by IHC has not been shown to predict response to tyrosine kinase inhibitor therapy in NSCLC [Meert, A. P., et al., Eur Respir J, 2002. 20(4): p. 975-81]. Thus, development of antibodies that specifically detect mutant EGFR protein and that may be used in IHC will be a valuable addition to the clinical diagnosis and treatment of lung cancer.
A related challenge facing diagnostic analysis of solid tumor samples including lung cancer tumors is access to the tissue sample. Repeated biopsies are not clinically feasible for almost all tumor types. Therefore, alternative sources of cancer cells must be obtained. This is especially important in the context of targeted therapeutics in which repeated tumor analysis may be used to guide the drug therapy. A number of cancer cell sources are available in some tumor types including circulating cancer cells (CTCs), ascites, bronchial swabs, ductal adenocarincoma is of a cancer tissue type selected from the group consisting of lung cancer, colon cancer, breast cancer, cervical cancer, pancreatic cancer, prostate cancer, stomach cancer, and esophageal cancer. circulating proteins may be detected by standard protein assays such as an ELISA assay. In this example, the mutation EGFR protein would be captured and detected with a pair of antibodies including an antibody against the total protein and an antibody to the mutation. Such an assay would enable routine and repeated analysis of treated patients to best match the choice of drug and drug regime to the direct affect the therapy was having on the patient's tumor.